Isolation and Characterization of the Promoter of the Human Prostate Cancer-specific DD3 Gene

Verhaegh, Gerald W.; Bokhoven, Adrie van; Smit, Frank; Schalken, Jack A.; Bussemakers, M. J. G.

doi:10.1074/jbc.m006293200

Cited by 36 publications

(40 citation statements)

References 29 publications

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“…One year later, the promoter was sequenced by Verhaegh et al 6 According to Gandini et al, 10 only the splicing variant of exon 4 is specific for prostate cancer. As PCA3 is highly expressed in prostate cancer compared to normal prostate and negative in other normal and malignant tissues, this molecule is regarded as a potential target for therapy 11,12 or an adjunct to diagnosis.…”

Section: Discussionmentioning

confidence: 99%

“…It is overexpressed in the majority of prostate cancers including distant metastases, expressed at a negligible level in nonmalignant prostate tissue and not expressed in other normal or malignant tissues. [6][7][8] As PCA3 is a noncoding gene, there is no corresponding protein eventually detectable by immunohistochemistry. 9 Also, the cellular location of the molecule has never been identified.…”

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confidence: 99%

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Identification of PCA3 (DD3) in prostatic carcinoma by in situ hybridization

et al. 2007

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PCA3 is a specific marker of prostatic carcinoma. However, PCA3 has been detected only at RNA level and a corresponding PCA3 protein has never been identified. The aim of this study was to develop a technique capable of detecting PCA3 RNA on histology sections and to assess the cellular location of the molecule. Fortyeight formalin-fixed paraffin-embedded blocks of prostatectomy specimens were selected for PCA3 detection by in situ hybridization by both radioactive and chromogenic methods. Of the 48 sections, 28 contained prostatic adenocarcinoma and 20 had benign tissue located distant from the tumor. Using the radioactive detection method, 26 of 28 available cases (93%) of cancers presented at least focal cytoplasmic PCA3 expression. The benign glands located in proximity of the cancer presented PCA3 expression in eight (29%) cases, whereas those situated distant to the tumor showed focal expression in 2 of 20 (10%) cases only. Highgrade prostatic intraepithelial neoplasia (HGPIN) expressed PCA3 in 25 of 26 (96%) cases. With the chromogenic detection method, 22 of the 24 interpretable cases (92%) of cancers had at least focal cytoplasmic staining. Benign glands located close to neoplastic glands expressed PCA3 in 8 (33%) cases, but none of those distant to the tumor expressed the marker. HGPIN was positive in 17 of 24 (71%) cases. The sensitivity, specificity, positive predictive value and negative predictive value for the detection of cancer were 93, 79, 71 and 95% for the radioactive detective method and 92, 80, 71 and 95% for the chromogenic detection method, respectively. Our study shows that PCA3 RNA is expressed by most prostate cancers and HGPIN. Normal glands rarely express the marker, except those located in immediate proximity of neoplastic glands, suggesting the presence of precursor molecular changes.

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Section: Discussionmentioning

confidence: 99%

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Identification of PCA3 (DD3) in prostatic carcinoma by in situ hybridization

et al. 2007

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“…The DD3 promoter was a generous gift from Dr. Verhaegh. 25 The fragment no. 1.12 ( À 152 to +62 bp) from their publication was used.…”

Section: Reporter Constructsmentioning

confidence: 99%

A novel method for the determination of basal gene expression of tissue-specific promoters: An analysis of prostate-specific promoters

Poel¹,

McCadden²,

Verhaegh

et al. 2001

Cancer Gene Ther

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Because the toxicity of suicide gene therapeutics is directly related to basal promoter activity, we developed an assay to test for promoter ''leakiness'' using a diphtheria toxin mutant. Sequences of 15 prostate -specific gene promoter constructs were cloned in an expression plasmid ( pBK; Stratagene, La Jolla, CA ) backbone driving expression of an attenuated mutant of diphtheria toxin A ( tox176 ). Low expression levels of the DT -tox176 result in significant protein synthesis inhibition reflected by a decreased expression of the luciferase activity of a simultaneously transfected CMV luciferase construct. ID 50 ( dose of plasmid with 50% luciferase inhibition ) was calculated for each promoter construct in different cell lines. Highest transactivational activity ( ID 50 < 75 ng ) was found for the CMV promoter in all cell lines, which is in agreement with the dual luciferase assay findings. Unlike the dual luciferase findings, however, the DT -tox176 assay showed protein inhibition of CN65 ( PSA promoter / enhancer ) and PSE -hK2 ( PSA enhancer and basal human kallikrein 2 promoter ) in HEK293 and DLD cells indicating ''leakiness'' of these promoter constructs. Low basal promoter activity in nonprostate cell lines was found for the minimal PSA promoter, hK2, DD3, and OC promoters. The DT -tox176 assay can better predict basal promoter activity compared to less sensitive dual luciferase assay. Cancer Gene Therapy ( 2001 ) 8, 927 -935

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“…DNA probes (0.5 ng) were incubated with nuclear extract (NE, 15 mg), and separated on 4% polyacrylamide gels as described (Verhaegh et al, 2000). For supershift assays, antibodies against Sp1 (4 mg, Santa Cruz), HIF-1a (2 mg, Santa Cruz), HIF-2a (6.3 mg, Novus Biologicals) or IgG control were added to the binding reactions.…”

Section: Electrophoretic Mobility Shift Assay (Emsa)mentioning

confidence: 99%

“…For each DNase-I footprint reaction, 1.5-3.0 fpu recombinant Sp1 protein (Promega) was used as described (Verhaegh et al, 2000).…”

Section: Dnase-i Footprint Analysismentioning

confidence: 99%

Strict regulation of CAIXG250/MN by HIF-1α in clear cell renal cell carcinoma

Grabmaier¹,

Weijert²,

Verhaegh³

et al. 2004

Oncogene

166

118

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Renal cell carcinoma of the clear cell type (ccRCC) is associated with loss of functional von Hippel-Lindau (VHL) protein and high, homogeneous expression of the G250 MN protein, an isoenzyme of the carbonic anhydrase family. High expression of G250MN is found in all ccRCCs, but not in most normal tissues, including normal human kidney. We specifically studied the mechanism of transcriptional regulation of the CAIX G250 gene in RCC. Previous studies identified Sp1 and hypoxia-inducible factor (HIF) as main regulatory transcription factors of G250 MN in various non-RCC backgrounds. However, G250MN regulation in RCC has not been studied and may be differently regulated in view of the HIF accumulation under normoxic conditions due to VHL mutations. Transient transfection of different G250 MN promoter constructs revealed strong promoter activity in G250 MN -positive RCC cell lines, but no activity in G250 MN -negative cell lines. DNase-I footprint and bandshift analysis demonstrated that Sp1 and HIF-1a proteins in nuclear extracts of RCC cells bind to the CAIX promoter and mutations in the most proximal Sp1 binding element or HIF binding element completely abolished CAIX promoter activity, indicating their critical importance for the activation of G250 expression in RCC. A close correlation between HIF-1a expression and G250 MN expression was observed. In contrast, no relationship between HIF-2a expression and G250 MN was seen. The participation of cofactor CBP/p300 in the regulation of G250 transcription was shown. In conclusion, HIF-1a and Sp1, in combination with CBP/p300, are crucial elements for G250 MN expression in ccRCC, and CAIX G250 can be regarded as a unique HIF-1a target gene in ccRCC.

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Isolation and Characterization of the Promoter of the Human Prostate Cancer-specific DD3 Gene

Cited by 36 publications

References 29 publications

Identification of PCA3 (DD3) in prostatic carcinoma by in situ hybridization

Identification of PCA3 (DD3) in prostatic carcinoma by in situ hybridization

A novel method for the determination of basal gene expression of tissue-specific promoters: An analysis of prostate-specific promoters

Strict regulation of CAIXG250/MN by HIF-1α in clear cell renal cell carcinoma

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